Malignant gliomas are the most common primary brain tumors and are characterized by invasive growth and recalcitrance to current therapies. Among glioma cell types, multipotent CD133+ cells have the capacity to initiate disease in immunodeficient mice (Singh et al., 2004). The identification of a tumor-initiating cell type in brain tumors suggests that, like hematological malignancies, the cellular heterogeneity of gliomas represents a hierarchical arrangement of differentiation states (Clarke et al., 2006). According to this paradigm, elimination of tumor-initiating cells may provide the greatest therapeutic benefit (Park et al., 2009). A primary impediment to investigating this fundamental concept in glioma biology is the lack of cell markers for characterizing the phenotypic heterogeneity of glioma tumor-initiating cells. Our background data in a primary xenotransplantation model suggest that CD133 expression encompasses discrete subpopulations of glioma cells with different biological characteristics. To study the cellular heterogeneity encompassed by CD133 expression, we propose to generate monoclonal variable lymphocyte receptor (VLR) antibodies for glioma progenitor cells by immunizing lampreys with CD133+ cells isolated from malignant glioma subtypes. The lamprey antibodies, which are structurally different proteins than our antibodies, will be analyzed for their potential to identify tumor-initiating cells and their lineages in a direct orthotopic xenotransplantation model. Proof-of-principle studies demonstrate that recombinant monoclonal VLR antibodies can be made against human tumor cells representative of different stages in B lymphocyte differentiation. We postulate that among other advantages unique to the VLR antibodies of jawless vertebrates, cellular epitopes otherwise impermissible to a mammalian antibody repertoire, because of self-tolerance, will be accessible to lamprey antibodies (Herrin et al., 2008). PUBLIC HEALTH RELEVANCE: Malignant gliomas are recalcitrant to current therapies and patients with this disease currently have a poor prognosis. The identification of cell types responsible for tumor growth and recurrence may lead to the development of more effective therapies. In this proposal a novel method, the adaptive immune system of jawless vertebrates, will be used to identify glioma tumor-initiating cells and their lineages in a direct orthotopic xenotransplantation model.